1. Field of Invention
The invention relates to the degradation of the hydrocarbon constituents of crude petroleum and petroleum products in any environment. It particularly relates to the degradation by emulsification, solubilization and break down of said petroleum materials by application of an effective amount of Actinomucor elegans or its enzymatic active principles.
2. Description of the Prior Art
There has been considerable effort expended in attempts to develop mechanical, chemical and microbiological processes for cleaning up oil leaks, spills and slicks on the world's land mass, oceans and seaways, which have become a common, virtually daily-occurring problem caused by sinking or damaged ships and broken oil pipelines. An additional extensive and related effort has been made to avoid and rectify oil pollution resulting from the cleaning of the tanks of oil tankers, bilges and fuel bunkers after the unloading of oil cargo or emptying of fuel stores. The pollution caused by the cleaning of petroleum storage tanks on tankers is particularly substantial, as several thousand gallons of crude petroleum may remain on the interior sides of the tanks after unloading of the cargo, which eventually forms a highly viscous, gel-like mass over time if the tanks are not cleaned. This mandates that the tanks be cleaned frequently, which results in the production of thousands of gallons of oil/water/cleaning compound for each vessel, which may initially be held in a slop tank, but eventually is dumped into the open water. While the cleaning compound normally constitutes one or a combination of the known biodegradable detergent compounds or compositions, there still is a substantial fouling problem and pollution from the petroleum material even though not aggravated by detergent pollution.
The seriousness of open sea oil pollution is known both from practical experience and from the pollution studies of Milz & Frazer, Jour. Petr. Techn. 24:255-262 (1972). Milz and Frazer found that an oil slick of 40 gallons of crude oil would cover a 200.times.30 foot area 10 minutes after being spilled into the open sea, which slick would expand to an area 100 feet wide and one-half mile long after one hour.
Despite this work and the vast literature reporting it, the art still lacks an efficient yet simple solution to the problem of cleaning up spills of crude petroleum and petroleum products. Known methods are cumbersome and of unknown efficacy in a given situation. Stein et al. German Patent No. 2547742 discloses a typical system, in which polymers are used to absorb crude petroleum in an oil slick, where the oil must be then removed by skimming of the spill-polluted water environment.
Particular emphasis has been placed in this area on the development of a microbial agent which demonstrates hydrocarbon degradation or consumptive properties, which properties essentially comprise the ability to emulsify, solubilize, break down and consume the deleterious petroleum materials. The normally water-soluble components of crude petroleum constitute only about 0.02% by weight of the material; it is known that bacteria capable of degrading hydrocarbons utilize them only in dissolved states. Hence most of the hydrocarbons present in crude petroleum, which are highly water insoluble, cannot be broken down by known microbial degradation techniques.
Experimental studies using crude petroleum as a microbial substrate have heretofore been carried out which have, however, led to the discovery of microbes having hydrocarbon degrading properties, as well as an affinity to the substrate, under laboratory conditions. Ludvik et al., Experientia 24:255 (1958), described a hydrocarbon-degrading yeast having cell components which made them adhere to oil droplets. Korowitz et al., J. Appl. Microbiology 30:10-19 (1975), disclosed strain UP-2, and showed the importance of the relationship of the size of the oil droplet to the growth of the microbe, that size being regulated by the emulsifying characteristics of the microbe while growing on the hydrocarbons. Iguchi et al., Agri Biol. Chem. 33:1657-58 (1969), showed that Candida petrophilium, which degraded hydrocarbons, produced an emulsifying agent composed of peptides and fatty acid moieties. Similar work with Arthrobacter, Brevibactorium and Nocardia species found that a trehalose-lipid was produced in the oil phase. Gholsen et al., U.S. Nat. Tech. Int. Serv. AD Rep. No. 757071 (1973), stated that, while a procedure to chemically modify enzymes in order to cause them to adhere to a hydrocarbon-water interface without appreciable loss of activity had been effective to some extent in a lysozome, RN-ase and 2 lipases, it was left to the future to provide microorganisms which would by production of extracellular emulsifying enzymes biodegrade hydrocarbons in oil spills.
The production by the known laboratory-effective microorganisms of emulsifying agents and subsequent droplet formation appears to enhance pseudosolubilization of the hydrocarbons and the uptake of those hydrocarbons into cells. Scott et al., J. Bacteriol. 127:469-480 (1976) showed that, with cells of Acinetobacter sp., the uptake of solid phase hydrocarbons was by pseudosolubilization. It is probable that the organism had evolved surface active substances which acted as wetting agents and as detergents.
The art has long discussed the potential advantages of artificially seeding polluted areas with pure or mixed cultures of microorganisms which are known to degrade the hydrocarbon constituents of crude petroleum and petroleum products. Efficacy demonstrated by experimental or industrial fermentor work using hydrocarbons, however, rarely has but the slightest relationship to what happens in the open sea or other large water or land environment. The regulated temperature and pH conditions, with optimum nutrition, aeration and agitation, of the fermentor or experimental set-up is totally absent in the natural environment or the sea. The art has also found such seeding to be impractical with the few known hydrocarbon-degrading microorganisms due to the impossibility of providing the nitrogenous and phosphoric nutrition necessary to sustain the microbes in such vast, open environments.
Solution of the nutritional problem by attempts to induce the oil degradation microbes to fix atmospheric nitrogen by recombinant techniques using bacterium-carrying plasmids for nitrogen fixation has been carried out (Gutnik et al. Ann, Rev. Appl. Microb. 370-396 (1977)), but has demonstrated no present success.
In sum, the art totally lacks any effective method, compound or composition capable of degrading crude petroleum and petroleum products in actual, non-laboratory conditions and environments, such as open ocean spills of crude petroleum. The art particularly lacks any such degradative method, compound or composition utilizing a microorganism such as a bacterium or fungus having such actual environment effectiveness.
There is a need in the art, then, for a method, compound and composition capable of effecting the degradation of crude petroleum and petroleum products in an environment, particularly a salt water environment, such as would be encountered in land or sea-borne spills of crude petroleum or petroleum products. There is a particular need for a microorganism-based method, compound and composition which demonstrates the capability of degrading and breaking down by emulsification, solubilization and ingestion of the hydrocarbon constituents of crude petroleum and petroleum products large quantities of such materials in any environment, including the catastrophic spill of crude petroleum which occurs in the sinking of an ocean-going tanker vessel and in the blowing out of an oil pipeline. There is a further need for a method, compound and composition which may be utilized to clean the interior of vessel tanks, particularly those of oil tankers used to transport crude petroleum and petroleum products. Particularly needed is a method, compound and composition which may be utilized in conjunction with the known biodegradable detergent compounds and compositions which the art now uses to carry out such cleaning.
The optimum combination of properties for a method, compound and composition for effecting the degradation of crude petroleum and petroleum products in an environment particularly one based in the utilization of a microorganism, is such that:
(1) the method, compound and composition must demonstrate acceptable efficacy in actual, non-laboratory environments, including inhospitable climates and various land and open water--salt water/ocean conditions, yet be in such form as to itself be non-toxic and non-deleterious, and to generate no deleterious products or chemicals harmful to or befouling of said environment; PA0 (2) the method, compound and composition must be longacting and rapid in the onset of its initial activity, and require no further support or sustaining activities after initiation and/or application to the crude petroleum or petroleum product; PA0 (3) the method, compound and composition must be self-sustaining, so as to require no additional provision for nutrients or other supporting chemicals or compositions other than what the crude petroleum or petroleum products provide; PA0 (4) the method, compound and composition, together with the degraded, solubilized crude petroleum or petroleum products, must be self-dissipating, so as to require no retrieval and disposal of any petroleum-laden component or material; PA0 (5) the method, compound and composition must be easy to effect and manufacture, while safe to personnel applying the composition or carrying out the process at all stages and times of its preparation and use; and PA0 (6) the method, compound and composition must be compatible with and capable of use in conjunction with known biodegradable detergent compounds and compositions.
None of the microorganism-based laboratory degradative processes or compositions known to the art, however, and particularly none of the actual environment methods or compositions (of which there are none which are microorganism based), provide this optimum combination of properties desirable with respect to the degradation of crude petroleum and petroleum products.